I want to count bacteria from biofilm with DNA probes and study bacterial interactions in biofilm.
I'm having some troubles doing the same thing with Candida biofilms. I´ve tried sonication but it did not worked very well.
Sonication tends to be very effective. We generally use a sonication with a probe, followed by serial dilution to determine CFU's I'll try to get the settings and post that later. Cooling on ice is important as the ultrasonic waves heat up the sample..
Thanks Bastiaan. I think that would be a solution, but my concern is how consistent the results are among many samples by using sonication probe.
We have obtained very reproducible results. The only concern is with very porous materials such as hydroxyapatite. CFU plating in general is time consuming.
Candida biofilms might be difficult in general to dislodge especially if you have a lot of hyphae. For removal of biofilms from well-plates we have used a cell-scraper commonly used by cell biologists. This worked well.
My experience with Sonication is best in terns of biofilom removal.
Hello, I got good results using two successive baths of sonnication. First 10 min with buffer at 4 ° C. We recovered the middle and then second with the same protocol. Finally, you can mark the plate with DAPI to verify that everything has been correctly removed from the plate.
Thanks, Everyone. What sonication power and frequency do you use for good results?
Sonication was performed on a device Branson sonicator B2501, the frequency was 40kHz and 230 W
Hello, has anyone checked that after sonification, how many bacteria are still alive?
I am working with E. coli biofilms. I grow them in plastic petri plates for 24 or 48 h. Remove the planktonic cells, wash the plate gently and simply scrape off the attached cells with a spreader or cell scrapper by putting some buffer or medium on the plate. It works well if you want to harvest the cells for counting, fluorescence measurement or for any reason where they need to be viable. Sonication is likely to kill the cells. As correctly pointed out by Davide, it is important to characterize the setup to ensure that the cells are alive. I would suggest to use sonication bath. If you are growing biofilms in microtiter plates, you can wash the wells gently, put some buffer or medium, seal the plate thoroughly by parafilm and put it in the sonication bath. Pulses of 30 to 60 s will detach the cells from the surface. Viability can be checked by plating. As far as my experience says, the cells are mostly viable.
Hi Pierre Lembré, May I ask what do you do exactly in the middle "recover" step? Do you Vortex or pipette in addition to sonication? Also, what kind of buffer do you use? Thanks
Could you please confirm whether we can use sonication for detaching bacteria associated to microalgae at same conditions as mentioned above by Mr. Pierre?
Vihang Ghalsasi Do you know how to extract the biofilm from glass vials for SEM imaging? Currently im working with homemade mouthwash. I would like to test the efficacy of removing biofilm
I am trying to attach bacteria on ligands immobilized on glass surface. As a control I have the bare glass surface. According to the above answers, if I sonicate the surface, will I be able to detach from glass while the ligand-attached cells remain intact?
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